Kinases
Kinases are a ubiquitous group of enzymes that catalyze the phosphoryl group transfer reaction from a phosphate donor (usually ATP) to a receptor substrate. Although all kinases catalyze essentially the same phosphoryl transfer reaction, they display remarkable diversity in their substrate specificity, structure, and the pathways in which they participate. A recent classification of all available kinase sequences (approximately 60,000 sequences) indicates that kinases may be grouped into 25 families of homologous proteins. These kinase families are assembled into 12 fold groups based on similarity of structural fold. 22 of the 25 families (approximately 98.8% of all sequences) belong to 10 fold groups for which the structural fold is known. Of the other 3 families, polyphosphate kinase forms a distinct fold group; the 2 remaining families are both integral membrane kinases and comprise the final fold group. These fold groups not only include some of the most widely spread protein folds, such as Rossmann-like fold, ferredoxin-like fold, TIM-barrel fold, and antiparallel β-barrel fold, but also all major classes (all α, all β, α+β, α/β) of protein structures. Within a fold group, the core of the nucleotide-binding domain of each family has the same architecture, and the topology of the protein core is either identical or related by circular permutation. Homology between the families within a fold group is not implied.
Group I (23,124 sequences) kinases incorporate protein S/T-Y kinase, atypical protein kinase, lipid kinase, and ATP grasp enzymes and further comprise the protein S/T-Y kinase, and atypical protein kinase family (22,074 sequences). These kinases include: choline kinase (EC 2.7.1.32); protein kinase (EC 2.7.137); phosphorylase kinase (EC 2.7.1.38); homoserine kinase (EC 2.7.1.39); 1-phosphatidylinositol 4-kinase (EC 2.7.1.67); streptomycin 6-kinase (EC 2.7.1.72); ethanolamine kinase (EC 2.7.1.82); streptomycin 3′-kinase (EC 2.7.1.87); kanamycin kinase (EC 2.7.1.95); 5-methylthioribose kinase (EC 2.7.1.100); viomycin kinase (EC 2.7.1.103); [hydroxymethylglutaryl-CoA reductase (NADPH2)] kinase (EC 2.7.1.109); protein-tyrosine kinase (EC 2.7.1.112); [isocitrate dehydrogenase (NADP+)] kinase (EC 2.7.1.116); [myosin light-chain] kinase (EC 2.7.1.117); hygromycin-B kinase (EC 2.7.1.119); calcium/calmodulin-dependent protein kinase (EC 2.7.1.123); rhodopsin kinase (EC 2.7.1.125); [beta-adrenergic-receptor] kinase (EC 2.7.1.126); [myosin heavy-chain] kinase (EC 2.7.1.129); [Tau protein] kinase (EC 2.7.1.135); macrolide 2′-kinase (EC 2.7.1.136); 1-phosphatidylinositol 3-kinase (EC 2.7.1.137); [RNA-polymerase]-subunit kinase (EC 2.7.1.141); phosphatidylinositol-4,5-bisphosphate 3-kinase (EC 2.7.1.153); and phosphatidylinositol-4-phosphate 3-kinase (EC 2.7.1.154). Group I further comprises the lipid kinase family (321 sequences). These kinases include: I-phosphatidylinositol-4-phosphate 5-kinase (EC 2.7.1.68); I D-myo-inositol-triphosphate 3-kinase (EC 2.7.1.127); inositol-tetrakisphosphate 5-kinase (EC 2.7.1.140); 1-phosphatidylinositol-5-phosphate 4-kinase (EC 2.7.1.149); 1-phosphatidylinositol-3-phosphate 5-kinase (EC 2.7.1.150); inositol-polyphosphate multikinase (EC 2.7.1.151); and inositol-hexakiphosphate kinase (EC 2.7.4.21). Group I further comprises the ATP-grasp kinases (729 sequences), which include inositol-tetrakisphosphate I-kinase (EC 2.7.1.134); pyruvate, phosphate dikinase (EC 2.7.9.1); and pyruvate, water dikinase (EC 2.7.9.2).
Group II (17,071 sequences) kinases incorporate the Rossman-like kinases. Group II comprises: (i) the P-loop kinase family (7,732 sequences), which include gluconokinase (EC 2.7.1.12); phosphoribulokinase (EC 2.7.1.19); thymidine kinase (EC 2.7.1.21); ribosylnicotinamide kinase (EC 2.7.1.22); dephospho-CoA kinase (EC 2.7.1.24); adenylylsulfate kinase (EC 2.7.1.25); pantothenate kinase (EC 2.7.1.33); protein kinase (bacterial) (EC 2.7.1.37); uridine kinase (EC 2.7.1.48); shikimate kinase (EC 2.7.1.71); deoxycytidine kinase (EC 2.7.1.74); deoxyadenosine kinase (EC 2.7.1.76); polynucleotide 5′-hydroxyl-kinase (EC 2.7.1.78); 6-phosphofructo-2-kinase (EC 2.7.1.105); deoxyguanosine kinase (EC 2.7.1.113); tetraacyldisaccharide 4′-kinase (EC 2.7.1.130); deoxynucleoside kinase (EC 2.7.1.145); adenosylcobinamide kinase (EC 2.7.1.156); polyphosphate kinase (EC 2.7.4.1); phosphomevalonate kinase (EC 2.7.4.2); adenylate kinase (EC 2.7.4.3); nucleoside-phosphate kinase (EC 2.7.4.4); guanylate kinase (EC 2.7.4.8); thymidylate kinase (EC 2.7.4.9); nucleoside-triphosphate-adenylate kinase (EC 2.7.4.10); (deoxy)nucleoside-phosphate kinase (EC 2.7.4.13); cytidylate kinase (EC 2.7.4.14); and uridylate kinase (EC 2.7.4.-); (ii) the phosphoenolpyruvate carboxykinase family (815 sequences), which includes protein kinase (HPr kinase/phosphatase) (EC 2.7.1.37); phosphoenolpyruvate carboxykinase (GTP) (EC 4.1.1.32); and phosphoenolpyruvate carboxykinase (ATP) (EC 4.1.1.49); (iii) the phosphoglycerate kinase (1,351 sequences) family, which includes phosphoglycerate kinase (EC 2.7.2.3) and phosphoglycerate kinase (GTP) (EC 2.7.2.10); (IV) the aspartokinase family (2,171 sequences), which includes carbamate kinase (EC 2.7.2.2); aspartate kinase (EC 2.7.2.4); acetylglutamate kinase (EC 2.7.2.8 1); glutamate 5-kinase (EC 2.7.2.1) and uridylate kinase (EC 2.7.4.-); (v) the phosphofructokinase-like kinase family (1,998 sequences), which includes 6-phosphofrutokinase (EC 2.7.1.1 1); NAD(+) kinase (EC 2.7.1.23); 1-phosphofructokinase (EC 2.7.1.56); diphosphate-fructose-6-phosphate I-phosphotransferase (EC 2.7.1.90); sphinganine kinase (EC 2.7.1.91); diacylglycerol kinase (EC 2.7.1.107); and ceramide kinase (EC 2.7.1.138); (vi) the ribokinase-like family (2,722 sequences), which includes glucokinase (EC 2.7.1.2); ketohexokinase (EC 2.7.1.3); fructokinase (EC 2.7.1.4); 6-phosphofructokinase (EC 2.7.1. 11); ribokinase (EC 2.7.1.15); adenosine kinase (EC 2.7.1.20); pyridoxal kinase (EC 2.7.1.35); 2-dehydro-3-deoxygluconokinase (EC 2.7.1.45); hydroxymethylpyrimidine kinase (EC 2.7.1.49); hydroxyethylthiazole kinase (EC 2.7.1.50); 1-phosphofructokinase (EC 2.7.1.56); inosine kinase (EC 2.7.1.73); 5-dehydro-2-deoxygluconokinase (EC 2.7.1.92); tagatose-6-phosphate kinase (EC 2.7.1.144); ADP-dependent phosphofructokinase (EC 2.7.1.146); ADP-dependent glucokinase (EC 2.7.1.147); and phosphomethylpyrimidine kinase (EC 2.7.4.7); (vii) the thiamin pyrophosphokinase family (175 sequences), which includes thiamin pyrophosphokinase (EC 2.7.6.2); and (viii) the glycerate kinase family (107 sequences), which includes glycerate kinase (EC 2.7.1.31).
Group III kinases (10,973 sequences) comprise (i) the ferredoxin-like fold kinases; (ii) the nucleoside-diphosphate kinase family (923 sequences), which includes nucleoside-diphosphate kinase (EC 2.7.4.6); (iii) the HPPK kinase family (609 sequences), which includes 2-amino-4-hydroxy-6-hydroxymethyldihydropteridine pyrophosphokinase (EC 2.7.6.3); (IV) the guanido kinase family (324 sequences), which includes guanidoacetate kinase (EC 2.7.3.1); creatine kinase (EC 2.7.3.2); arginine kinase (EC 2.7.3.3); and lombricine kinase (EC 2.7.3.5); (v) the histidine kinase family (9,117 sequences), which includes protein kinase (histidine kinase) (EC 2.7.1.37); [pyruvate dehydrogenase(lipoamide)] kinase (EC 2.7.1.99); and [3-methyl-2-oxybutanoate dehydrogenase(lipoamide)] kinase (EC 2.7.1.115).
Group IV kinases (2,768 sequences) incorporate H-like kinases, which include hexokinase (EC 2.7.1.1); glucokinase (EC 2.7.1.2); fructokinase (EC 2.7.1.4); rhamnulokinase (EC 2.7.1.5); mannokinase (EC 2.7.1.7); gluconokinase (EC 2.7.1.12); L-ribulokinase (EC 2.7.1.16); xylulokinase (EC 2.7.1.17); erythritol kinase (EC 2.7.1.27); glycerol kinase (EC 2.7.1.30); pantothenate kinase (EC 2.7.1.33); D-ribulokinase (EC 2.7.1.47); L-fucolokinase (EC 2.7.1.51); L-xylulokinase (EC 2.7.1.53); allose kinase (EC 2.7.1.55); 2-dehydro-3-deoxygalactonokinase (EC 2.7.1.58); N-acetylglucosamine kinase (EC 2.7.1.59); N-acylmannosamine kinase (EC 2.7.1.60); polyphosphate-glucose phosphotransferase (EC 2.7.1.63); beta-glucoside kinase (EC 2.7.1.85); acetate kinase (EC 2.7.2.1); butyrate kinase (EC 2.7.2.7); branched-chain-fatty-acid kinase (EC 2.7.2.14); and propionate kinase (EC 2.7.2.-).
Group V kinases (1,119 sequences) incorporate TIM β/α-barrel kinases, which include pyruvate kinase (EC 2.7.1.40).
Group VI kinases (885 sequences) incorporate GHMP kinases. These enzymes include galactokinase (EC 2.7.1.6); mevalonate kinase (EC 2.7.1.36); homoserine kinase (EC 2.7.1.39); L-arabinokinase (EC 2.7.1.46); fucokinase (EC 2.7.1.52); shikimate kinase (EC 2.7.1.71); 4-(cytidine 5′-diphospho)-2-C-methyl-D-erythriol kinase (EC 2.7.1.148); and phosphomevalonate kinase (EC 2.7.4.2).
Group VII kinases (1,843 sequences) incorporate AIR synthetase-like kinases, which include thiamine-phosphate kinase (EC 2.7.4.16) and selenide, water dikinase (EC 2.7.9.3).
Group VIII kinases (565 sequences) incorporate riboflavin kinases (565 sequences), which include riboflavin kinase (EC 2.7.1.26).
Group IX kinases (197 sequences) incorporate dihydroxyacetone kinases, which include glycerone kinase (EC 2.7.1.29).
Group X kinases (148 sequences) incorporate putative glycerate kinases, which include glycerate kinase (EC 2.7.1.31).
Group XI kinases (446 sequences) incorporate polyphosphate kinases, which include polyphosphate kinases (EC 2.7.4.1).
Group XII kinases (263 sequences) incorporate integral membrane kinases. Group XII comprises the dolichol kinase family, which include dolichol kinases (EC 2.7.1.108); and the undecaprenol kinase family, which include undecaprenol kinases (EC 2.7.1.66).
Kinases, which are among the best-studied enzymes at the structural, biochemical, and cellular levels, play indispensable roles in numerous cellular metabolic and signaling pathways. Even though all kinases use the same phosphate donor (in most cases, ATP) and appear to catalyze apparently the same phosphoryl transfer reaction, they display remarkable diversity in their structural folds and substrate recognition mechanisms. This is probably due largely to the extraordinarily diverse nature of the structures and properties of their substrates.
Signal Transduction Pathways
The AGC family of protein kinases, which comprise isoforms of protein kinase B (PKB, also known as Akt), p70 ribosomal S6 kinase (S6K), serum- and glucocorticoid-induced protein kinase (SGK), and atypical isoforms of protein kinase C (PKC), are activated within minutes of insulin- or growth factor induced stimulation of phosphatidylinositol 3-kinase (PI3-kinase). Once activated, PKB/Akt phosphorylates and modulates the function of a number of important regulatory proteins, resulting in inhibition of apoptosis, formation of cell division and stimulation of glucose uptake and storage. The serine/threonine kinase Akt (protein kinase B) is a critical enzyme in signal transduction pathways involved in cell proliferation, apoptosis, angiogenesis, and diabetes. Three isoforms of Akt (α, β, γ or Akt 1, 2, 3) are known in mammals. These isoforms exhibit a high degree of homology but differ slightly in the localization of their regulatory phosphorylation sites. The Akt enzymes are composed of three functionally distinct regions: 1) an N-terminal pleckstrin homology (PH) domain; 2) a central catalytic domain; and 3) a C-terminal hydrophobic motif. The PH domain in the N-terminal region of Akt provides a lipid binding module to direct Akt to PIP2 (phosphatidyl inositol bisphosphate or the products obtained by cleavage of PIP3) and PIP3 (phosphatidyl inositol (3,4,5)-triphosphate, the product of the class I phosphoinositide 3-kinase activity on phosphatidyl inositol (4,5)-bisphosphate), interacts with 3′-phosphoinositides and helps to recruit Akt to the plasma membrane.
In unstimulated cells, Akt is constitutively phosphorylated at Ser124, in the region between the PH and catalytic domains, and on Thr450, in the C-terminal region (in Aktα). Activation of Akt involves growth factor binding to a receptor tyrosine kinase and activation of PI 3-K (PI 3-K phosphorylates membrane bound PIP2 to generate PIP3). The binding of PIP3 to the PH domain anchors Akt to the plasma membrane and allows its phosphorylation and activation by PDK1 (pyruvate dehydrogenase kinase isozyme 1). Akt is fully activated following its phosphorylation at two regulatory residues, a threonine residue on the kinase domain and a serine residue on the hydrophobic motif. These motifs are structurally and functionally conserved within the AGC kinase family. Phosphorylation at Thr308 and Ser473 is required for the activation of Aktα. Phosphorylation at Thr309 and Ser474 activates Aktβ. Phosphorylation at Thr305 activates Aktγ. Akt activation requires phosphorylation of a threonine residue on the kinase domain, catalyzed by PDK1. This causes a charge-induced conformational change, and allows substrate binding and an increased rate of catalysis. Phosphorylation at the serine residue, primarily by mTOR/richtor complex (mTORC2), augments Akt activity by approximately 10-fold. Studies indicate that DNA-PK and PKCbII phosphorylate the serine residue on the regulatory subunit. The hydrophobic motif of Akt, without threonine phosphorylation, is more susceptible to the action of phosphatases; however, the dually phosphorylated and fully active enzyme is stable, allowing its localization to the nucleus and other sites. The activity of Akt is negatively regulated by PTEN (phosphatase and tensin homolog gene whose product acts as a phosphatase to dephosphorylate phosphatidylinositol (3,4,5)-triphosphate) and SHIP(SH2-containing inositol phosphatase, INPP5D).
Akt facilitates growth factor-mediated cell survival and blocks apoptotic cell death by deactivating (via phosphorylation) pro-apoptotic factors such as Bad, caspase-9, and Forkhead transcription factors (AFX, Daf-16, FKHR). The phosphorylation of Bad at Ser136 promotes its association with 14-3-3 proteins in the cytosol; this prevents Bad from localizing at the mitochondria to induce apoptosis. Akt promotes cell survival by inactivating caspase-9 via phosphorylation at Ser196. Similarly, activated Akt phosphorylates Forkhead family members, resulting in their sequestration in the cytoplasm. In the absence of survival factors and Akt activity, Forkhead family members translocate to the nucleus, wherein they initiate a program of gene expression (for example, FasL) that promotes cell death. Akt also phosphorylates and activates IKKα (a subunit of 1κB alpha kinase complex that has an important role in the activation of nuclear factor-κB (NF-κB), a key regulator of normal and tumor cell proliferation, apoptosis and response to chemotherapy) at Thr23. The activated IKKα, in turn, phosphorylates IκB, targeting it for ubiquitination and proteasomal degradation. This leads to the activation and nuclear translocation of NF-κB, and the transcription of NF-kB-dependent pro-survival genes which include Bcl-xL and caspase inhibitors.
Akt also phosphorylates and inactivates GSK-3 (glycogen synthase kinase-3), allowing the activation of glycogen synthase to proceed. GSK-3 phosphorylates cyclin D (a regulator of G1 to S phase transition), targeting cyclin D for proteolysis. Thus, the inactivation of GSK-3 may promote the up-regulation of cyclin D and enhance cell cycling.
Studies indicate Akt phosphorylates Chk1 (a DNA damage effector kinase) at Ser280 thereby preventing human protein kinases ATM (ataxiaAtaxia telangiectasia mutated) and ATR (Ataxia telangiectasia and Rad3 related) from activating Chk1 via phosphorylation at Ser345. This may be of therapeutic significance as Chk1 inhibition enhances sensitization of tumors to chemotherapeutic agents.
Akt phosphorylates Cdc25B on Ser353, resulting in its cytoplasmic accumulation. Cdc25B undergoes activation during S-phase and has a role in activating the mitotic kinase Cdk1/cyclin B in the cytoplasm. This relocation of Cdc25B to the cytoplasm allows Akt to regulate Cdc25B function and participate in controlling the entry of cells into mitosis.
The regulation of Akt by a number of upstream oncogenes and tumor suppressor genes influences cancer progression. Breast cancer cell lines express Aktα in varying degrees. The Akt inhibitor, 1L-6-hydroxymethylchiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate, reduces survival of both drug resistant and drug sensitive multiple myeloma cells. Akt also has a critical role in tumorigenesis. Akt is activated when tumor suppressors, such as cell cycle inhibitor p27 and PTEN, lose their functions. Akt impairs the nuclear import of p27 by phosphorylation of p27 at Thr157. Cytoplasmic mislocalization of p27 has been strongly linked to loss of differentiation and poor outcome in breast cancer. Akt also is reported to associate physically with endogenous p21 (a cell cycle inhibitor). The phosphorylation of p21 at Thr145 by Akt causes p21 localization to the cytoplasm and subsequent degradation.
Akt and p53 (also known as protein 53 or tumor protein 53, a transcription factor that regulates the cell cycle) have opposing roles in signaling pathways that determine cell survival. Under conditions where the apoptotic effect of p53 is dominant, destruction of Akt has a role in accelerating the apoptotic process. In apoptosis-prone cells, p53-dependent signaling enables downregulation of Akt, which predisposes cells to rapid apoptosis in response to stress signals. Under certain circumstances, Akt activation may overcome the death promoting effects of p53 and may rescue cells from apoptosis. Studies indicate that Akt can phosphorylate Mdm2 (a protein encoded by an oncogene that modulates p53 tumor suppressor activity) on Ser166 and Ser188 and promote Mdm2 translocation to the nucleus wherein Mdm2 destabilizes p53 and enhances its degradation via the proteasomal pathway.
Kinase Inhibition
The eukaryotic protein kinases constitute one of the largest superfamilies of homologous proteins that are related to each other by virtue of their catalytic domains. Most related protein kinases are specific for either serine/threonine phosphorylation or tyrosine phosphorylation. Stimulation of protein kinases is considered to be one of these enzymes most common activation mechanisms in signal transduction systems and therefore plays an integral role in the cellular response to extracellular stimuli. Many substrates are known to undergo phosphorylation by multiple protein kinases. A considerable amount of information on primary sequence of the catalytic domains of various protein kinases has been published. These sequences share a large number of residues involved in ATP binding, catalysis, and maintenance of structural integrity. Most protein kinases possess a well conserved 30-32 kDa catalytic domain. Studies have attempted to identify and utilize regulatory elements of protein kinases. These regulatory elements include antibodies, blocking peptides, and inhibitors.
Inhibitors
Enzyme inhibitors are molecules that bind to enzymes thereby decreasing enzyme activity. The binding of an inhibitor may stop substrate from entering the active site of the enzyme and/or hinder the enzyme from catalyzing its reaction. Inhibitor binding is either reversible or irreversible. Irreversible inhibitors usually react with the enzyme and change it chemically, for example, by modifying key amino acid residues needed for enzymatic activity. In contrast, reversible inhibitors bind non-covalently and produce different types of inhibition depending on whether these inhibitors bind the enzyme, the enzyme-substrate complex, or both.
Enzyme inhibitors often are evaluated by their specificity and potency. The term “specificity” as used herein refers to the selective attachment or influence of one substance on another. The term “potency” as used herein refers to efficacy, effectiveness, strength, or, typically, the dissociation constant, which indicates the concentration needed to inhibit an enzyme.
Inhibitors of protein kinases have been studied for use as tools in protein kinase activity regulation Inhibitors have been studied for use with, for example, cyclin-dependent (Cdk) kinase, MAP kinase, serine/threonine kinase, Src Family protein tyrosine kinase, tyrosine kinase, calmodulin (CaM) kinase, casein kinase, checkpoint kinase (Chk1), GSK-3, JNK, MEK, myosin light chain kinase (MLCK), protein kinase A, Akt (protein kinase B), protein kinase C, protein kinase G, protein tyrosine kinase, Raf kinase, and Rho kinase.
Antibodies
Antibodies (or “immunoglobulins”) are gamma globulin proteins produced by B lymphocytes of the immune system in response to an antigen used by the body to identify and neutralize foreign objects having that antigen. In their native form, they are typically made of basic structural units—each with two large heavy (H) chains and two small light (L) chains—to form, for example, monomers with one unit, dimers with two units or pentamers with five units. There are several different types of antibody heavy chains, and several different kinds of antibodies, which are grouped into different isotypes based on which heavy chain they possess. Five different antibody isotypes are known in mammals, which perform different roles, and help direct the appropriate immune response for each different type of foreign object they encounter. The specificity and binding affinity of an antibody are dictated by the three polyvariable loops of the VL chain and the three hypervariable loops of the VH chain located on each arm of that antibody. Variations in the lengths and sequences of these loops define the antibody-combining site (ACS). As used herein, the term “antibody” includes, by way of example, both naturally occurring and non-naturally occurring antibodies. Specifically, the term “antibody” includes polyclonal antibodies and monoclonal antibodies, and fragments thereof. Furthermore, the term “antibody” includes chimeric antibodies and wholly synthetic antibodies, and fragments thereof. The terms “epitope” and “antigenic determinant” are used interchangeably herein to refer to the site on a molecule that an ACS recognizes and to which that antibody binds/attaches itself. An epitope may be an antigenic determinant/antigen binding site on a kinase inhibiting peptide. The epitope may be primary, secondary, or tertiary-sequence related.
The specificity of the interactions between certain antibodies and protein kinases has been studied for use in protein kinase activity regulation. Antibodies have been isolated for use with, for example, MAP kinase pathways, protein kinase A, protein kinase B, protein kinase G, serine/threonine kinases, glycogen-synthase kinase-3 (GSK-3), stress-activated protein (SAP) kinase pathways, and tyrosine kinases. Additionally, antibodies have been isolated for use with protein kinase inhibitors and protein kinase substrates.
Blocking Peptides
A peptide is a chemical compound that is composed of a chain of two or more amino acids; the carboxyl group of one amino acid is linked to the amino group of an adjacent amino acid to form a peptide bond. The term “polypeptide” is used herein in its broadest sense to refer to a sequence of subunit amino acids, amino acid analogs or peptidomimetics, wherein the subunits are linked by peptide bonds. The peptides or polypeptides may by chemically synthesized or expressed recombinantly. Peptides have been used in the study of protein structure and function. Synthetic peptides may be used as probes to see where protein-peptide interactions occur. Inhibitory peptides may be used in clinical research to examine the effects of peptides on the inhibition of protein kinases, cancer proteins and other disorders.
The use of several blocking peptides has been studied. For example, extracellular signal-regulated kinase (ERK), a MAPK protein kinase (meaning any of a group of protein serine/threonine kinases that respond to extracellular stimuli (antigens) and regulate various cellular activities), is essential for cellular proliferation and differentiation. The activation of MAPKs requires a cascade mechanism whereby MAPK is phosphorylated by an upstream MAPKK (MEK) which is then, in turn phosphorylated by a third kinase MAPKKK (MEKK). This inhibitory peptide functions as a MEK decoy by binding to ERK. It contains the amino-terminal 13 amino acids (GMPKKKPTPIQLN) [SEQ ID NO: 149] of MEK1 and binds to ERK. This blocks ERK activation by MEK as ERK is unable to interact with MEK. The ERK inhibitory peptide also contains a protein transduction (PTD) sequence (DRQIKIWFQNRRMKWKK) [SEQ ID NO: 150] derived from Antennapedia which renders the peptide cell permeable.
Other blocking peptides include autocamtide-2 related inhibitory peptide (AIP). This synthetic peptide is a highly specific and potent inhibitor of Ca2+/calmodulin-dependent protein kinase II (CaMKII). AIP is a non-phosphorylatable analog of autocamtide-2, a highly selective peptide substrate for CaMKII. AIP inhibits CaMKII with an IC50 of 100 nM (IC50 is a concentration of the inhibitor required to obtain 50% inhibition). The AIP inhibition is non-competitive with respect to syntide-2 (CaMKII Peptide Substrate) and ATP but competitive with respect to autocamtide-2. The inhibition is unaffected by the presence or absence of Ca2+/calmodulin. CaMKII activity is completely inhibited by 1 μM AIP; PKA, PKC and CaMKIV are not affected. The amino acid sequence of AIP is: KKALRRQEAVDAL (Lys-Lys-Ala-Leu-Arg-Arg-Gln-Glu-Ala-Val-Asp-Ala-Leu) [SEQ ID NO: 151].
Other blocking peptides include cyclin-dependent kinase 5 (Cdk5) inhibitory peptide (CIP). Cdk5 phosphorylates tau at Alzheimer's Disease (AD)-specific phospho-epitopes when it associates with the p25 regulatory component. p25 is a truncated activator of the Cdk-p25 heterodimer (a microtubule associated protein), which is produced from the physiological Cdk5 activator p35 upon exposure to amyloid-beta (Aβ, a protein implicated in AD) peptides. Upon neuronal infections with CIP, CIPs selectively inhibit p25/Cdk5 activity and suppress the aberrant tau phosphorylation in cortical neurons. The reasons for the specificity demonstrated by CIP are not fully understood.
Additional blocking peptides have been studied for ERK2, ERK3, p38/HOG1, protein kinase C, casein kinase II, Ca2+/calmodulin kinase IV, casein kinase II, Cdk4, Cdk5, DNA-PK, PAK3, PI-3 kinase, PI-5 kinase, PSTAIRE, ribosomal S6 kinase, GSK-4, GCK, SAPK, SEK1, and FAK.
Protein Transduction Domains
New drug delivery technologies occupy an important niche in treatments as they enable drugs to be more effective. Drug delivery still is considered a poor relation to drug discovery, with greater than 95% of all new potential therapeutics having poor pharmacokinetics. The greatest impediment for cytosolic release of therapeutic molecules is the membrane barrier of target cells. Protein transduction domains (PTDs), also referred to as Trojan peptides, membrane translocating sequences or cell permeable proteins, are a class of peptides that are generally capable of penetrating the plasma membrane of mammalian cells. PTDs are generally 10-16 amino acids in length and may be grouped according to their composition, for example peptides rich in arginine and/or lysine. PTDs also may be used to assist novel HSP27 kinase inhibitors to penetrate cell membranes (see, e.g., PCT/US2007/16246, filed Jul. 16, 2007, entitled “Polypeptic Inhibitors of HSP27 and Uses Thereof,” which is incorporated by reference herein in its entirety). PTDs are capable of transporting compounds of many types and molecular weights across mammalian cells. These compounds include effector molecules, such as proteins, DNA, conjugated peptides, oligonucleotides, and small particles such as liposomes. When PTDs are chemically linked or fused to other proteins these fusion proteins are still able to penetrate the plasma membrane and enter cells. Although the exact mechanism of this transduction is unknown, internalization of these proteins is not believed to be receptor-mediated or transporter-mediated. The use of PTDs capable of transporting effector molecules into cells has become increasingly attractive in the design of drugs as they promote the cellular uptake of cargo molecules. These cell penetrating peptides, generally categorized as amphipathic or cationic depending on their sequence, provide a non-invasive delivery technology for macromolecules.
Viral PTD Containing Proteins
The first proteins to be described as having transduction properties were viral in origin. These proteins still are the most commonly accepted models for PTD action. The HIV-1 Transactivator of transcription (TAT) and HSV-1 VP 22 protein are the best characterized viral PTD containing proteins.
TAT (HIV-1 trans-activator gene product) is an 86-amino acid polypeptide that act as a powerful transcription factor of the integrated HIV-1 genome. TAT stimulates viral replication in latently infected cells. The translocation properties of the TAT protein enable it to activate quiescent infected cells and may be involved in priming of uninfected cells for subsequent infection by regulating many cellular genes, including cytokines. The minimal PTD of TAT is the 9 amino acid protein sequence RKKRRQRRR (TAT49-57) [SEQ ID NO: 152]. Studies utilizing a longer fragment of TAT demonstrated successful transduction of fusion proteins up to 120 kDa. The addition of multiple TAT-PTDs and synthetic TAT derivatives have been demonstrated to mediate membrane translocation. TAT PTD containing fusion proteins have been used as therapeutic moieties in experiments involving cancer, for transporting a death-protein into cells, and in disease models of neurodegenerative disorders.
VP22 is the HSV-1 tegument protein, a structural part of the HSV virion. VP22 is capable of receptor independent translocation and accumulates in the nucleus. This property of VP22 classifies the protein as a PTD containing peptide. Fusion proteins comprising full length VP22 have been efficiently translocated across the plasma membrane.
Homeoproteins with Intercellular Translocation Properties
Homeoproteins are highly conserved, transactivating transcription factors involved in morphological processes. They bind to DNA through a specific sequence of 60 amino acids. The DNA-binding homeodomain is the most highly conserved sequence of the homeoprotein. Several homeoproteins exhibit PTD like activity; they are capable of efficient translocation across cell membranes in an energy-independent and endocytosis-independent manner without cell type specificity.
The Antennapedia protein (Antp) is a trans-activating factor capable of translocation across cell membranes; the minimal sequence capable of translocation is a 16 amino acid peptide corresponding to the third helix of the protein's homeodomain. The internalization of this helix occurs at 4° C. suggesting that this process is not endocytosis dependent. Peptides of up to 100 amino acids produced as fusion proteins with AntpHD penetrate cell membranes.
Other homeodomains capable of translocation include Fushi tarazu (Ftz) and Engrailed (En) homeodomain. Since the third helix of all homeodomains is highly conserved, it is likely that other homeodomains may possess similar characteristics.
Synthetic PTDs
Several PTD peptides have been synthesized. Many of these synthetic peptides are based on existing and well documented peptides, while others are selected for their basic residues and/or positive charge, which are thought to be crucial for PTD function. These synthetic peptides include: PTD-4 (YARAAARQARA) [SEQ ID NO: 153]; PTD-5 (RRQRRTSKLMKR) [SEQ ID NO: 154]; MST-1 (AAVLLPVLLAAR) [SEQ ID NO: 155]; L-R9 (RRRRRRRRR) [SEQ ID NO: 156]; and Peptide 2 (SGWFRRWKK) [SEQ ID NO: 157].
Human PTDs
Human PTDs may circumvent potential immunogenicity issues when used as a therapeutic upon introduction into a human patient. Peptides with PTD sequences include: Hoxa-5, Hox-A4, Hox-B5, Hox-B6, Hox-B7, HOX-D3, GAX, MOX-2, and FtzPTD, all of which share the sequence found in AntpPTD (RQIKIWFQNRRMKWKK) [SEQ ID NO: 158]. Other PTDs include Islet-1, interleukin-1β, tumor necrosis factor, and the hydrophobic sequence from Kaposi-FGF (K-FGF or FGF-4) signal peptide which is capable of energy-, receptor-, and endocytosis-independent translocation. Unconfirmed PTDs include members of the Fibroblast Growth Factor (FGF) family.
At present, it is possible to produce a given protein molecule by recombinant DNA technology for in vivo therapeutic applications. Although small molecules or peptides capable of crossing cellular membranes have been successfully designed to deliver small or moderately large proteins, it remains a challenge to deliver the recombinant proteins to desired targets in vivo. Despite developments in the area of protein transduction peptides, the classical delivery methods of protein-coding genes via adeno-associated virus, adenovirus, lentivirus, herpes virus vectors, and plasmid expression vectors remain the preferred choice for expression of proteins.
Viral vector-mediated gene expression is considered the most efficient and reliable approach for expressing functional proteins de novo in mitotically active or postmitotically blocked cell types due to their natural abilities to deliver the specific genes to permissive cells. Nonetheless, viral vectors invariably are required in large doses to achieve therapeutic expression levels of intended proteins and may integrate with the host chromatin material. Because these properties may have undesirable consequences for host genetic systems, safety remains a serious concern for their ultimate clinical application.
While an alternative approach, i.e., to produce recombinant proteins exogenously and then deliver them systemically or by localized injections into the target organs, appears to have a better safety profile, the delivery and bioavailability of recombinant proteins into cells or tissues needs refinement.
Several studies have shown the potential of PTD in drug discovery and transduction of proteins up to 120 kDa into different cells. In vivo injection of fusion proteins systemically has demonstrated the effectiveness of the PTD in protein delivery. Despite successful applications, questions about potency of PTD mediated protein transduction still remain unsolved. Further, some studies in PTD-mediated fusion protein transduction in vitro/in vivo and attempting to induce an immune response have failed. Further, intracellular expression of PTD fusion proteins or other non-secretory proteins may not achieve the same biodistribution as that of recombinant protein. Further, entry of PTD through the blood-brain barrier remains elusive.
The delivery of a diverse set of cargo ranging from small molecules to particulate cargo has been attempted using different types of cell penetrating peptides in vitro and in vivo. However, the internalization mechanism of these peptides is an unresolved issue to date, with dramatic changes in view regarding the involvement of an energy dependent process involving endocytosis as a pathway of internalization. An improvment in the effectiveness of PTDs would significantly increase bioavailability and lower the required doses of existing and novel therapeutics.
The present invention provides transduction domain peptides that are useful for the inhibition of kinases. The present invention further provides a class of peptides that include certain transduction domains that are useful as inhibitors of kinase activity. The present invention further provides transduction domain peptides that are useful as therapeutic agents for a variety of hyper plastic and neoplastic disorders. The present invention further provides transduction domain peptides that are useful as substances to cause cell death.